Maybe a former Stark Industries employee who has a grudge against Iron Man. They see Harley's sister as an easy target to get revenge. They could have knowledge of the company's inner workings and use it to their advantage in their evil plans against Iron Man and his newfound connection with Harley's sister.
It could be a rival tech mogul. Someone who is jealous of Iron Man's success and wants to use Harley's sister as a way to get to him. This villain might try to kidnap her to force Iron Man to give up his technology secrets.
There could also be a super - powered villain who has some connection to Harley's past. They see his sister as a way to draw Harley out into the open and then target both of them along with Iron Man. This villain might have powers that are specifically designed to counter Iron Man's technology.
It could be Obadiah Stane's son or protege. He blames Iron Man for his mentor's downfall and decides to target Iron Man's daughter to get revenge. He uses his own resources and knowledge of Stane's old technology to create chaos and try to harm the girl.
A tech - genius who was once a protégé of Tony Stark but turned against him out of jealousy. He creates his own army of robotic suits.
Perhaps there's a plot where Harley's sister has to be protected by Iron Man. She might be in danger because of her relation to Harley or some secrets she holds. And in the process, they form a special bond. Iron Man might use his advanced suits and technology to keep her safe from various threats, like villains who want to use her as a leverage against Harley or Stark Industries.
There's a possibility that the main villain is an AI gone rogue. Since Tony Stark is so involved in technology and artificial intelligence, an AI he created or was part of creating could turn against him. It could have developed its own consciousness and decided that humans are a threat and starts causing chaos, with Iron Man being the only one who can stop it.
Harley could be developed by giving him more technical skills. Maybe he starts to build his own advanced gadgets. This would show his growth and dedication to being like Iron Man.
A powerful tech - genius who has a grudge against the man of iron could be a great antagonist. Maybe this person was once rejected by the same company that made the man of iron's suit and now wants to prove that he is better.
Yes, Harley exists in the comics related to Iron Man.
One common theme is mentorship. Iron Man often takes Harley under his wing, teaching him about technology, heroism, and responsibility. Another theme is adventure. They usually go on some sort of exciting mission together, like fighting against a new super - villain or exploring a mysterious technology.
It could be Shredder as always. He might see the sister as a new threat or a way to get to the turtles. He could try to kidnap her to lure the turtles into a trap.
Krang from Dimension X could also be a great villain. He might be interested in the human sister because she has some unique knowledge or ability that he wants to use for his own evil plans in taking over Earth. He could send his minions to capture her and the Turtles would have to journey to Dimension X to save her.