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Antonio (from the sequel) will return.
It's pretty likely that there'll be a third Despicable Me, since there's already a spin-off in the works. Considering his plotline got dropped after he started flirting with another girl and he's the son of the (defeated) villain, he may be back to avenge his father, and to get a defeat by Margo's hands, for breaking her heart.

Perkins (Bank of Evil) and Ramsbottom (AVL) are brothers.
And, like the Duke brothers, are playing a high-stakes game. How many monuments can Bank od Evil-backed villains steal vs. how many supervillains can AVL agents arrest. The bet? TWO dollars. Because the stakes on this game are higher.

The villains from the first film DID get their comeuppance.
Miss Hattie's orphanage was probably shut down due to her cruel conditions and the kids were put in another orphanage or found homes. Mr. Perkins probably went mad after his son was stuck on the moon and he suffered a nervous breakdown.

Margo and Antonio had a fight.
Yes, Antonio was flirting with another girl. But! Maybe he and Margo regarding Gru. Antonio started making fun of Gru and even called him a "second rate father" and other such names. Margo tried to play it off, but then went full-on ballistic at Antonio for his bad mouthing of her dad right in front of him. The two then broke up. Antonio, angry at the whole thing, decided to make Margo jealous. He started dancing with another girl...in Margo's direction.

Edith is the daughter of the samurai Mifune.
Blonde hair? Check. Aversion to romance? Check. Badass McNinja? Freaking check.

The Avery we see Margo texting is a girl.
But Margo still has a crush on her.

Kyle was neutered between movies
Before he was massively aggressive attacking everything on sight. Now not only does he not attack anything, but he looks completely depressed in a "I don't give a crap anymore" kind of vibe. Makes sense because Gru wouldn't want Kyle hurting the girls once they settled in as a real family.

The Despicable Me villians are Girl Genius Sparks.
First, note that we never see a villain with inherent powers (with the possible exception of El Macho), only Gadgeteer Geniuses. This also explains why Gru went into the jams business and generally doesn't seem to do as much inventing after the girls: inventing gadgets affects his brain and he doesn't like that effect anymore.

Agnes's sound ability may have come from accidentally ingesting a green potion that Edith dropped from the first movie.
We all know Gru was, at best, neglectful of the girls and he even treated them like pets at first. Agnes was okay with eating candy from a pet bowl, what makes her not want to eat the bright green fluid on the floor when Edith accidentally dropped?

The super serum is a mutated form of Rage.
Testing on animals? Check. Makes the infected go crazy? Check.

Kyle was a product of PX-41
Wild, excessive hair? Check. Large spiky teeth? Check. Massive agressiveness? Check.

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