The Shredder could be a great main villain. He's always been a big threat to the Turtles and could pose an even greater danger to the cubs and the lone rat.
Maybe an old rival of the lone rat from his past could emerge as the main villain. This rival could be jealous of the rat's new family with the cubs and wants to take them away or cause them harm.
A new, more powerful mutant created by some evil scientist could be the main villain. This mutant could have abilities that are specifically designed to counter the lone rat's skills and the cubs' natural defenses.
The main character is obviously the lone rat. He is likely a key figure in the story, guiding and protecting the cubs.
One idea could be exploring the relationship between the lone rat and the cubs in more depth. Maybe show how the lone rat teaches the cubs survival skills.
Another plot could be that the lone rat discovers a long - lost relative of the cubs. This relative could bring both hope and trouble. The relative might have knowledge that could help the cubs grow stronger, but also attract unwanted attention from the rat's old enemies.
Sasuke could also be an interesting choice. He is always seeking power, and a rat contract could offer him new ways to gain information about his enemies. He could use the rats' stealth abilities to his advantage in his quest for revenge.
The Foot Clan could be a main antagonist. They might see the baby sister as a way to get to the turtles, either by kidnapping her or using her to blackmail them. Another could be Shredder himself if he returns. He would surely not pass up the opportunity to use the baby sister against the turtles. And Baxter Stockman could also be an antagonist. He might be interested in using the baby sister for some of his mad - scientist experiments.
Perhaps a new character could be the main antagonist in this fanfiction. A power - hungry alchemist who discovers the Turtles' mutation and wants to use their baby forms to create an army of mutant slaves. This alchemist would stop at nothing to get his hands on the baby Turtles and experiment on them.
It could be a powerful and mysterious figure from the shadows. Maybe a rogue huntsman gone bad who has his own agenda against the lone huntsman. This antagonist might have a vendetta for some past event.
Maybe a corrupt businessman who discovers some ancient technology from the Old West that gives him superhuman abilities. He wants to use these abilities to take over Gotham and expand his business empire at any cost. He sees Batman as a threat to his plans and when the Lone Ranger shows up, he tries to get rid of them both. His greed and new - found powers make him a formidable foe for the two heroes.
Splinter might be the main helper. He has raised the Turtles, so he has experience with taking care of young ones. He could teach the other Turtles the proper way to hold baby Mikey, how to soothe him when he cries, and give them advice on how to keep him clean and healthy. Splinter's wisdom would be really important in this situation.
Perhaps it's an old acquaintance of Splinter from his past. Someone who he knew before he became the turtles' master. This person could have some history with Splinter and their reunion could be filled with emotions and revelations. They would be more likely to trust each other and work towards Splinter's well - being.